Behavioural response to SKF 38393 and quinpirole following chronic antidepressant treatment

The effects of chronic administration of antidepressant drugs (21-22 days s.c. via osmotic mini-pumps) on the behavioural responses of male Sprague-Dawley rats to (-)-quinpirole hydrochloride (0.05 mg kg-1 s.c., 5 min) and (+/-)-SKF 38393 hydrochloride (10 mg kg-1 s.c., 5 min) were investigated. Desipramine hydrochloride (10 mg kg-1 per day), phenelzine sulphate (10 mg kg-1 per day) and clorgyline hydrochloride (1 mg kg-1 per day) attenuated the suppression of locomotor activity induced by quinpirole, a dopamine D2-like receptor agonist, while clomipramine hydrochloride (10 mg kg-1 per day) was without effect. Yawning elicited by quinpirole was absent in phenelzine- and clorgyline-treated rats, but unaffected in rats treated chronically with desipramine and clomipramine. SKF 38393, a dopamine D1-like receptor agonist, significantly increased locomotor activity and time spent grooming in control animals. There were no significant effects of antidepressants on the behavioural responses to SKF 38393.     

Subunit-specific interactions of cyanide with the N-methyl-D-aspartate receptor

Cyanide can potentiate N-methyl-D-aspartate receptor-mediated physiological responses in neurons. Here we show that this phenomenon may be attributable to a subunit-specific chemical modification of the receptor directly by the toxin. N-Methyl-D-aspartate (30 microM)-induced whole cell responses in mature (22-29 days in vitro) rat cortical neurons were potentiated nearly 2-fold by a 3-5-min treatment with 2 mM potassium cyanide, as did a similar treatment with 4 mM dithiothreitol. A 1-min incubation with the thiol oxidant 5,5'-dithiobis(2-nitrobenzoic acid) (0.5 mM) readily reversed the potentiation induced by either cyanide or dithiothreitol. Cyanide did not increase further currents previously potentiated by dithiothreitol nor was it able to potentiate responses during brief co-application with the agonist. Transient expression studies in Chinese hamster ovary cells with wild-type and mutated recombinant N-methyl-D-aspartate subunits (NR) demonstrated that cyanide selectively potentiated NR1/NR2A receptors, presumably via the chemical reduction of NR2A. In contrast, currents mediated by NR1/NR2B receptors were somewhat diminished by the metabolic inhibitor. Some of the effects of cyanide on NR1/NR2B receptors may be mediated by the formation of a thiocyanate adduct with a cysteine residue located in NR1. Cyanide thus is able to distinguish chemically between two different N-methyl-D-aspartate receptor subtypes and produce diametrically opposing functional effects.     

Enhanced sensitivity of the MRL/MpJ mouse to the neuroplastic and behavioral effects of acute and chronic antidepressant treatments.

Adult hippocampal neurogenesis has been implicated in the pathophysiology of depression and in the therapeutic effects of antidepressant drugs. Current immunohistochemical methods that study neurogenesis are time consuming and labor intensive. Therefore, a significantly more rapid flow cytometric method was characterized to measure neurogenesis in the adult mouse brain. The sensitivity of mice to the effects of antidepressant treatments is dependent on genetic background. Thus, studies were conducted comparing the responsiveness of 2 inbred mouse strains, MRL/MpJ and C57BL/6J, to the acute and chronic effects of antidepressants on neurochemistry and behavior. Acutely, MRL/MpJ mice displayed more robust behavioral and neurochemical responses to pharmacologically distinct antidepressants than C57BL/6J mice. Chronic administration of the antidepressant drugs fluoxetine and desipramine produced robust elevations in hippocampal cell proliferation and brain-derived neurotrophic factor (BDNF) protein levels in MRL/MpJ mice. C57BL/6J mice treated similarly with antidepressant drugs were mainly unresponsive on these measures. Mice were tested in the novelty-induced hypophagia (NIH) paradigm to examine a behavioral response associated with chronic, but not acute, antidepressant treatment. Only MRL/MpJ mice were behaviorally responsive to chronic antidepressant administration in the NIH paradigm. The positive effects of chronic antidepressants on hippocampal cell proliferation and BDNF paralleled the ability of these drugs to produce changes in NIH behavior. These studies highlight the advantages of using flow cytometry to study hippocampal neurogenesis and identify the MRL/MpJ mouse as a strain with superior response to antidepressant drug treatments that may lead to a better understanding of the genetics behind antidepressant efficacy and sensitivity. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Modulation of N-methyl-D-aspartate receptor function by glycine transport

The recent discovery of glycine transporters in both the central nervous system and the periphery suggests that glycine transport may be critical to N-methyl-D-aspartate receptor (NMDAR) function by controlling glycine concentration at the NMDAR modulatory glycine site. Data obtained from whole-cell patch-clamp recordings of hippocampal pyramidal neurons, in vitro, demonstrated that exogenous glycine and glycine transporter type 1 (GLYT1) antagonist selectively enhanced the amplitude of the NMDA component of a glutamatergic excitatory postsynaptic current. The effect was blocked by 2-amino-5-phosphonovaleric acid and 7-chloro-kynurenic acid but not by strychnine. Thus, the glycine-binding site was not saturated under the control conditions. Furthermore, GLYT1 antagonist enhanced NMDAR function during perfusion with medium containing 10 microM glycine, a concentration similar to that in the cerebrospinal fluid in vivo, thereby supporting the hypothesis that the GLYT1 maintains subsaturating concentration of glycine at synaptically activated NMDAR. The enhancement of NMDAR function by specific GLYT1 antagonism may be a feasible target for therapeutic agents directed toward diseases related to hypofunction of NMDAR.     

Structure-activity studies of conantokins as human N-methyl-D-aspartate receptor modulators

The activities of conantokin-G (con-G), conantokin-T (con-T), and several novel analogues have been studied using polyamine enhancement of [3H]MK-801 binding to human glutamate-N-methyl-D-aspartate (NMDA) receptors, and their structures have been examined using CD and 1H NMR spectroscopy. The potencies of con-G[A7], con-G, and con-T as noncompetitive inhibitors of spermine-enhanced [3H]MK-801 binding to NMDA receptor obtained from human brain tissue are similar to those obtained using rat brain tissue. The secondary structure and activity of con-G are found to be highly sensitive to amino acid substitution and modification. NMR chemical shift data indicate that con-G, con-G[D8, D17], and con-G[A7] have similar conformations in the presence of Ca2+. This consists of a helix for residues 2-16, which is kinked in the vicinity of Gla10. This is confirmed by 3D structure calculations on con-G[A7]. Restraining this helix in a linear form (i.e., con-G[A7,E10-K13]) results in a minor reduction in potency. Incorporation of a 7-10 salt-bridge replacement (con-G[K7-E10]) prevents helix formation in aqueous solution and produces a peptide with low potency. Peptides with the Leu5-Tyr5 substitution also have low potencies (con-G[Y5,A7] and con-G[Y5,K7]) indicating that Leu5 in con-G is important for full antagonist behavior. We have also shown that the Gla-Ala7 substitution increases potency, whereas the Gla-Lys7 substitution has no effect. Con-G and con-G[K7] both exhibit selectivity between NMDA subtypes from mid-frontal and superior temporal gyri, but not between sensorimotor and mid-frontal gyri. Asn8 and/or Asn17 appear to be important for the ability of con-G to function as an inhibitor of polyamine-stimulated [3H]MK-801 binding, but not in maintaining secondary structure. The presence of Ca2+ does not increase the potencies of con-G and con-T for NMDA receptors but does stabilize the helical structures of con-G, con-G[D8,D17], and, to a lesser extent, con-G[A7]. The NMR data support the existence of at least two independent Ca2+-chelating sites in con-G, one involving Gla7 and possibly Gla3 and the other likely to involve Gla10 and/or Gla14.     

The teratogenicity of N-methyl-D-aspartate (NMDA) receptor antagonists

While it is generally accepted that y-aminobutyric acid type A (GABA(A))-receptor agonists decrease anxiety by facilitating the neuronal influx of chloride, the site of action within the brain is not clearly delineated. To gain further insight into the locus of anxiolytic action of GABA in the brain, we measured the distribution of GABA-stimulated chloride influx in seven regions of the brain from high- and low-anxiety rats. Our results show a significant increase in GABA-gated chloride influx in the hypothalamus of rats exhibiting low anxiety. The role of the hypothalamus in the regulation of anxiety is briefly discussed.     

Modulation of the N-methyl-D-aspartate receptor by haloperidol: NR2B-specific interactions

The dopaminergic antagonist haloperidol has an eight- to 10-fold higher affinity for NMDA receptors containing the NR2B (epsilon2) subunit, showing the same subunit specificity as ifenprodil, polyamines, and magnesium. In the present study, we have compared the effects of mutations altering polyamine and ifenprodil sensitivity on haloperidol sensitivity of NMDA receptors. As seen for spermidine stimulation, high-affinity haloperidol inhibition is governed by the region around amino acid 198, based on results from chimeric murine NR2A/NR2B (epislon1/epsilon2) receptors. Mutation of epsilon2E201 in this region to asparagine or arginine causes a 10-fold decrease in the ability of haloperidol to inhibit 125I-MK-801 binding. Epsilon2E201 does not govern the interactions of ifenprodil, because all of the mutants at epsilon2E201 exhibited wild-type affinity for ifenprodil. Mutation of epsilon2R337 causes a 400-fold loss in apparent affinity for ifenprodil but does not change the effects of haloperidol. The structural determinants of spermidine stimulation do not perfectly match those for haloperidol inhibition, as mutations of E200 remove haloperidol inhibition but do not alter polyamine stimulation. The present results thus demonstrate that although spermidine, haloperidol, and ifenprodil share subunit selectivity and overlapping pharmacology, they also have specific structural determinants.     

Expression of mRNAs encoding subunits of the N-methyl-D-aspartate receptor in cultured cortical neurons

The expression of mRNAs encoding subunits of the N-methyl-D-aspartate (NMDA) receptor was examined in cortical neurons maintained in primary culture. Cultures were prepared from embryonic day 17 rat neocortex. At this developmental age, levels of NR1, NR2A, NR2B, and NR2C mRNA were low or undetectable. Expression of NR1 mRNA increased progressively between days 1 and 21 in vitro. The amount of NR2A mRNA did not change between days 1 and 7 but increased between days 7 and 21. In contrast, levels of NR2B mRNA increased between days 1 and 7, with little further change after day 7. The level of NR2B mRNA was approximately 4-fold higher than that of NR2A mRNA in 21-day cultures. Using ligand binding assays, the proportion of NMDA receptors having a low affinity for ifenprodil was also found to increase over time in culture. The increase in the expression of receptors having a low affinity for ifenprodil and the increase in NR1 and NR2A mRNAs were reduced or prevented by maintaining cells in medium with a low concentration of serum. The results are consistent with the hypothesis that inclusion of the NR2A subunit in native NMDA receptors is responsible for their low affinity for ifenprodil. Splice variants of NR1 lacking the 5' (amino-terminal) insert were found to be the predominant forms of NR1 in cultured neurons. Variants containing the 5' insert represented only a small (< or = 5%) fraction of total NR1 mRNA, and their proportion was not altered as a function of time in culture. Time-dependent changes in the properties of NMDA receptors and in the expression of subunit mRNA occurring in cultured neurons are similar to changes observed in developing rat brain. Thus, the developmental sequence of NMDA receptor expression that occurs in vivo is partially retained in neurons maintained in vitro.     

Neuroprotective activity of a new class of steroidal inhibitors of the N-methyl-D-aspartate receptor

Release of the excitatory neurotransmitter glutamate and the excessive stimulation of N-methyl-D-aspartate (NMDA)-type glutamate receptors is thought to be responsible for much of the neuronal death that occurs following focal hypoxia-ischemia in the central nervous system. Our laboratory has identified endogenous sulfated steroids that potentiate or inhibit NMDA-induced currents. Here we report that 3alpha-ol-5beta-pregnan-20-one hemisuccinate (3alpha5betaHS), a synthetic homologue of naturally occurring pregnanolone sulfate, inhibits NMDA-induced currents and cell death in primary cultures of rat hippocampal neurons. 3alpha5betaHS exhibits sedative, anticonvulsant, and analgesic properties consistent with an action at NMDA-type glutamate receptors. Intravenous administration of 3alpha5betaHS to rats (at a nonsedating dose) following focal cerebral ischemia induced by middle cerebral artery occlusion significantly reduces cortical and subcortical infarct size. The in vitro and in vivo neuroprotective effects of 3alpha5betaHS demonstrate that this steroid represents a new class of potentially useful therapeutic agents for the treatment of stroke and certain neurodegenerative diseases that involve over activation of NMDA receptors.     

Modulation of glucocorticoid receptor gene expression by antidepressant drugs

The literature on the setting mechanisms of dental amalgams made from powders of silver-rich alloys of tin and/or copper has been critically reviewed. In Part 1 of the review, the microstructure and phase content of recently set amalgams are described. The composition, morphology, and location of product phases are emphasized, since these features are clues to the setting reaction. Thus, Part 1 provides the background needed to understand the kinetics of the setting reactions, which is the topic of Part 2 of the review.     

Brain-derived neurotrophic factor rapidly enhances phosphorylation of the postsynaptic N-methyl-D-aspartate receptor subunit 1

Although neurotrophins have traditionally been regarded as neuronal survival factors, recent work has suggested a role for these factors in synaptic plasticity. In particular, brain-derived neurotrophic factor (BDNF) rapidly enhances synaptic transmission in hippocampal neurons through trkB receptor stimulation and postsynaptic phosphorylation mechanisms. Activation of trkB also modulates hippocampal long-term potentiation, in which postsynaptic N-methyl-D-aspartate glutamate receptors play a key role. However, the final common pathway through which BDNF increases postsynaptic responsiveness is unknown. We now report that BDNF, within 5 min of exposure, elicits a dose-dependent increase in phosphorylation of the N-methyl-D-aspartate receptor subunit 1. This acute effect occurred in hippocampal synaptoneurosomes, which contain pre- and postsynaptic elements, and in isolated hippocampal postsynaptic densities. Nerve growth factor, in contrast, caused no enhancement of phosphorylation. These results suggest a potential mechanism for trophin-induced potentiation of synaptic transmission.     

Differential profiles of binding of a radiolabeled agonist and antagonist at a glycine recognition domain on the N-methyl-D-aspartate receptor ionophore complex in rat brain

Addition of several polyamines, including spermidine and spermine, was effective in inhibiting binding of the antagonist ligand [3H]5,7-dichlorokynurenic acid ([3H]-DCKA) a Gly recognition domain on the N-methyl-D-aspartic acid (NMDA) receptor ionophore complex in rat brain synaptic membranes. In contrast, [3H]DCKA binding was significantly potentiated by addition of proposed polyamine antagonists, such as ifenprodil and (+/-)-alpha-(4-chlorophenyl)-4-[(4-fluorophenyl) methyl]-1-piperidine ethanol, with [3H]Gly binding being unchanged. The inhibition by spermidine was significantly prevented by inclusion of ifenprodil. In addition, spermidine significantly attenuated the abilities of four different antagonists at the Gly domain to displace [3H]DCKA binding virtually without affecting those of four different agonists. Phospholipases A2 and C and p-chloromercuribenzosulfonic acid were invariably effective in significantly inhibiting [3H]DCKA binding with [3H]Gly binding being unaltered. Moreover, the densities of [3H]DCKA binding were not significantly different from those of [3H]-Gly binding in the hippocampus and cerebral cortex, whereas the cerebellum had more than a fourfold higher density of [3H]Gly binding than of [3H]DCKA binding. These results suggest that the Gly domain may have at least two different forms based on the preference to agonists and antagonists in the rodent brain.     

Regulation of N-methyl-D-aspartate receptor function by constitutively active protein kinase C

The ability of the constitutively active fragment of protein kinase C (PKM) to modulate N-methyl-D-aspartate (NMDA)-activated currents in cultured mouse hippocampal neurons and acutely isolated CA1 hippocampal neurons from postnatal rats was studied using patch-clamp techniques. The responses of two heterodimeric combinations of recombinant NMDA receptors (NR1a/NR2A and NR1a/NR2B) expressed in human embryonic kidney 293 cells were also examined. Intracellular applications of PKM potentiated NMDA-evoked currents in cultured and isolated CA1 hippocampal neurons. This potentiation was observed in the absence or presence of extracellular Ca2+ and was prevented by the coapplication of the inhibitory peptide protein kinase inhibitor(19-36). Furthermore, the PKM-induced potentiation was not a consequence of a reduction in the sensitivity of the currents to voltage-dependent blockade by extracellular Mg2+. We also found different sensitivities of the responses of recombinant NMDA receptors to the intracellular application of PKM. Some potentiation was observed with the NR1a/NR2A subunits, but none was observed with the NR1a/NR2B combination. Applications of PKM to inside-out patches taken from cultured neurons increased the probability of channel opening without changing single-channel current amplitudes or channel open times. Thus, the activation of protein kinase C is associated with potentiation of NMDA receptor function in hippocampal neurons largely through an increase in the probability of channel opening.     

Circuit-specific alterations of N-methyl-D-aspartate receptor subunit 1 in the dentate gyrus of aged monkeys

Age-associated memory impairment occurs frequently in primates. Based on the established importance of both the perforant path and N-methyl-D-aspartate (NMDA) receptors in memory formation, we investigated the glutamate receptor distribution and immunofluorescence intensity within the dentate gyrus of juvenile, adult, and aged macaque monkeys with the combined use of subunit-specific antibodies and quantitative confocal laser scanning microscopy. Here we demonstrate that aged monkeys, compared to adult monkeys, exhibit a 30.6% decrease in the ratio of NMDA receptor subunit 1 (NMDAR1) immunofluorescence intensity within the distal dendrites of the dentate gyrus granule cells, which receive the perforant path input from the entorhinal cortex, relative to the proximal dendrites, which receive an intrinsic excitatory input from the dentate hilus. The intradendritic alteration in NMDAR1 immunofluorescence occurs without a similar alteration of non-NMDA receptor subunits. Further analyses using synaptophysin as a reflection of total synaptic density and microtubule-associated protein 2 as a dendritic structural marker demonstrated no significant difference in staining intensity or area across the molecular layer in aged animals compared to the younger animals. These findings suggest that, in aged monkeys, a circuit-specific alteration in the intradendritic concentration of NMDAR1 occurs without concomitant gross structural changes in dendritic morphology or a significant change in the total synaptic density across the molecular layer. This alteration in the NMDA receptor-mediated input to the hippocampus from the entorhinal cortex may represent a molecular/cellular substrate for age-associated memory impairments.     

Agmatine selectively blocks the N-methyl-D-aspartate subclass of glutamate receptor channels in rat hippocampal neurons

We investigated in rat hippocampus neurons whether 4-(aminobutyl)guanidine (agmatine), formed by decarboxylation of L-arginine by arginine decarboxylase and metabolized to urea and putrescine, can modulate the function of N-methyl-D-aspartate (NMDA) receptor channels. In cultured hippocampal neurons studied by whole-cell patch clamp, extracellular-applied agmatine produced a voltage- and concentration-dependent block of NMDA but not alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid nor kainate currents. Analysis of the voltage dependence of the block suggests that agmatine binds at a site located within the NMDA channel pore with a dissociation constant of 952 microM at 0 mV and an electric distance of 0.62. We also tested effects of several agmatine analogs. Arcaine (1,4-butyldiguanidine) also produced a similar voltage-dependent block of the NMDA current, whereas putrescine (1, 4-butyldiamine) had little effect, suggesting that the guanidine group of agmatine is the active moiety when blocking the NMDA channel. Moreover, spermine (an endogenous polyamine) potentiated the NMDA current even in the presence of blocker agmatine or arcaine, suggesting that the guanidine-containing compounds agmatine and arcaine interact with the NMDA channel at a binding site different from that of spermine. Our results indicate that in hippocampal neurons agmatine selectively modulates the NMDA subclass of glutamate receptor channels mediated by the interaction between the guanidine group and the channel pore. The results support other data that agmatine may function as an endogenous neurotransmitter/neuromodulator in brain.     

Interactions between N-methyl-D-aspartate receptor antagonists and the discriminative stimulus effects of morphine in rats

NMDA receptor antagonists have previously been reported to alter some pharmacological and behavioral effects of acute and chronic opioid administration. The present study assessed the interactions of NMDA antagonists with the discriminative stimulus properties of morphine. Adult male Long Evans rats were trained to discriminate 3.2 mg/kg of s.c. morphine from water under a two-lever fixed-ratio 10 schedule of food reinforcement. During test sessions. I.p. injections of the noncompetitive NMDA receptor antagonist dizocilpine (0.03-0.2 mg/kg), the competitive antagonists NPC 17742 (1-16 mg/kg), and SDZ 220-581 (0.1-3 mg/kg), the polyamine site antagonist eliprodil (3-17.3 mg/kg), the glycine-site partial agonist (+)-HA-966 (3-56 mg/kg), and the nonselective glutamate antagonist kynurenic acid (30-150 mg/kg) were coadministered with s.c. morphine (1-3.2 mg/kg; interaction tests) or water (generalization tests). In generalization tests, none of the compounds completely substituted for morphine. Concurrent administration of morphine and NMDA antagonists did not greatly alter the discriminative stimulus properties of morphine. Various doses of NPC 17742, SDZ 220-581, or (+)-HA-966 somewhat increased levels of morphine-appropriate lever selection, whereas some attenuation of morphine-lever selection was obtained when morphine was coadministered with eliprodil. These results show that NMDA antagonists have minimal interactions with the discriminative stimulus effects of morphine.     

Cloning and structure of the gene encoding the human N-methyl-D-aspartate receptor (NMDAR1)

The complete gene encoding the human N-methyl-D-aspartate receptor subunit NR1 (NMDAR1) has been isolated on a single cosmid clone. The gene is composed of 21 exons distributed over a total length of about 31 kb. More than 24 kb were sequenced. Exons 4, 20 and 21 are identical in their amino-acid sequence to those exons that are subject to alternative splicing in rat, indicating that all eight NMDAR1 isoforms found in rat will also be expressed in the human brain. Computer analysis of the pre-mRNA sequence revealed no secondary structures stable enough to explain alternative splicing. We suggest that cell-specific factors control expression of different isoforms. The promoter region contains two perfect copies of the recognition sequence for the Drosophila even-skipped protein, indicating that the developmentally regulated expression of NMDAR1 is controlled by a homeobox protein. The complete cosmid clone covering NMDAR1 was mapped to chromosome 9q34.3-qter by fluorescent in situ hybridization (FISH). The telomeric location is supported by an imperfect (CA)n repeat homologous to a subtelomeric repeat on chromosome 16p.